Project Summary: T cell immunotherapy is poised to be a major player in the future of medicine. The success of CD19 CAR T cells in acute lymphoblastic leukemia has been impressive and gives a glimpse of what the next generation of engineered T cells may be capable of. In the future, CAR-targeted T cells may be valuable immunotherapy for more than just cancer. In particular, targeted T regulatory cell therapy is a promising possibility for treatment of autoimmune conditions and tolerance induction in transplant recipients. Current methods of CAR T cell production are dependent on laborious isolation, modification, and expansion steps on a per-patient basis. This makes them highly expensive with little possibility of an off-the-shelf product. Induced pluripotent stem cells (iPSC) allow the reprogramming of adult somatic cells to a stem-cell like fate from patients with specific MHC haplotypes to overcome this issue. The combination of MHC-banked iPSCs and CAR technology sets the stage for a renewable and highly controllable off-the-shelf T cell therapeutic. However, efforts to differentiate iPSCs into T cells have struggled with the production of hematopoietic progenitors with robust T cell potential and a blockade in T cell maturation at the CD4/8 double positive stage. In this proposal we demonstrate the marked impact of modulating the NOTCH signaling pathway on T-capable progenitor output. We propose to combine this success with single-cell RNAseq to reveal the true identity of the T cell progenitor. This work will uncover key signaling pathways and answer lingering questions about the nature of iPSC- derived hematopoietic progenitors. It will also leverage improved efficiency in differentiation cultures. In addition, we propose to use temporally controlled expression of CARs with a range of antigen affinities during T cell differentiation to bypass their developmental blockade and drive maturation into functional T cells. These two synergistic but not dependent goals will be a vital and powerful step toward truly functional iPSC- derived T cells and would also open the door for exploring signals necessary to drive CD4 helper T cell commitment and T reg lineage specification.